Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice

Abstract

We tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD(+) intermediate, increases arterial SIRT1 activity and reverses age-associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium-dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)-mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age-associated impairment in EDD was restored in OC by the superoxide (O2-) scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s(-1) vs. 337 ± 3 cm s(-1) ) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic O2- production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen-I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO-mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s(-1) ) and EM (3694 ± 315 kPa), normalized O2- production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen-I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD(+) threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age-related arterial dysfunction by decreasing oxidative stress.

Keywords: NAD+; SIRT1; arterial stiffness; endothelial dysfunction; oxidative stress.

Figure 1

Endothelium‐dependent, nitric oxide (NO)‐dependent, and endothelium‐independent dilation. (A) Dose–responses to the endothelium‐dependent dilator acetylcholine (ACh) in young and old control (YC and OC) and young and old NMN‐supplemented (YNMN and ONMN) mice (n = 13–22 per group). (B) NO‐dependent dilation (Max DilationACh − Max DilationACh + l‐NAME) (n = 5–12 per group). (C) Dose–responses to the endothelium‐independent dilator sodium nitroprusside (SNP) (n = 7–22 per group). Values are mean ± SEM. *P < 0.05 vs. all.

Figure 2

Vascular oxidative stress. (A) Maximal dose–response to the endothelium‐dependent dilator acetylcholine (ACh) in young and old control (YC and OC) and young and old NMN‐supplemented (YNMN and ONMN) mice in the presence or absence of TEMPOL (n = 5–9 per group) *P < 0.05 vs. TEMPOL. (B) Superoxide production, assessed by electron paramagnetic resonance (EPR). Values are normalized to YC mean value. Representative EPR signal below (n = 4–9 per group). (C) Nitrotyrosine (NT) abundance in aorta. Data are expressed relative to α‐smooth muscle actin and normalized to YC mean value. Representative Western blot images below (n = 4–6 per group). Values are mean ± SEM. *P < 0.05 vs. all; # P < 0.05 vs. YC.

Figure 3

Large elastic artery stiffness. (A) Aortic pulse wave velocity (aPWV) in young and old control (YC and OC) and young and old NMN‐supplemented (YNMN and ONMN) mice (n = 5–9 per group). (B) Elastic modulus (n = 5–6 per group). (C) Total collagen 1 (Col 1) expression in aorta (n = 4–9 per group). (D) Total elastin expression in aorta. Values normalized to YC mean value. Representative images below (n = 4–11 per group). Values are mean ± SEM. Bars = 100 μm. *P < 0.05 vs. all; # P < 0.05 vs. YC.

Figure 4

SIRT1 expression and activity. (A) SIRT1 expression in aorta of young and old control (YC and OC) and young and old NMN‐supplemented (YNMN and ONMN) mice (n = 5–7 per group). (B) Ratio of acetylated to total NFκB in aorta (n = 5–11 per group). Data are expressed relative to α‐smooth muscle actin and normalized to YC mean value. Representative Western blot images below. Values are mean ± SEM. #P < 0.05 vs. YC; †P < 0.05 vs. OC.

Figure 5

NAD⁺ and MnSOD. (A) NAD⁺ production in aortas from young mice incubated in vitro with vehicle or NMN for 48 h (n = 3–5 per group). (B) Manganese superoxide dismutase (MnSOD) expression in aortas from old mice incubated in vitro with vehicle or NMN for 48 h (n = 4–5 per group). Values normalized to vehicle mean value. Representative images below. Values are mean ± SEM. Bars = 100 μm. *P < 0.05.